Bromodomains play a crucial role in regulating many cellular and physiological processes, and disruption of their function contributes to the etiology of multiple diseases. Specifically, the bromodomains in subfamily IV (BRD1/7/9, ATAD2, KIAA1240 and BRPF1/3) 1 are found in proteins associated with the development of acute myeloid leukemia, prostate cancer and breast cancer 2-4. Bromodomains are chromatin reader domains that recognize acetylated lysine 5, and function by linking their associated protein/enzyme complex to chromatin. Recently, several key studies demonstrated that the bromodomain is a druggable target, and small molecule inhibitors are currently in clinical trials for the treatment of cancer and hematological malignancies 6-8. Interest in developing therapeutics targeting the bromodomain has grown dramatically, and a computational study by Vidler et al., 2012 analyzed the druggability of human bromodomains based on structural classification 13. This study correctly predicted the druggability of the BET family of bromodomains, and suggested that bromodomains within family IV are potential new targets. However, while the structures of these bromodomains have been solved, their biological function(s) and how they recognize acetylated histone ligands is largely unknown. The overall objective of the research proposed here is to establish the molecular mechanism of acetylated histone ligand selection by the family IV bromodomains. Specifically, we plan to identify their histone ligands, and outline the specificity determinants of the bromodomain-histone interaction. Our central hypothesis is that the family IV bromodomains recognize a similar subset of histone acetyllysine marks, and utilize a uniquely conserved binding mode to select for their ligands. This proposal aims to: (1) identify the histone ligands of family IV bromodomains and (2) outline the molecular mechanism of histone recognition by family IV bromodomains. A unique combination of biochemical, biophysical and structural biology techniques will be used to characterize the structural and functional role of the family IV bromodomains. The histone tail ligands will be identified and verified using peptide array assays in combination with nuclear magnetic resonance (NMR) chemical shift perturbation techniques. The atomic resolution structures of the family IV bromodomains bound to their histone tail ligands will be solved by NMR or X-ray crystallography. Site-directed mutagenesis coupled with NMR and ITC will be used to measure the effects on ligand binding and clarify the role of ordered waters on acetyllysine coordination. Our results will reveal the atomic details of ligand coordination, and outline the rules driving acetyllysine recognition and histone binding. This research will fundamentally advance our understanding of how bromodomains decipher the histone code to regulate critical cellular functions including signal transduction, gene expression and chromatin remodeling. The results will provide the high-resolution molecular details critical for the development of new therapeutics.